WO2024085226A1 - Kit de détection et procédé de détection de molécule cible - Google Patents

Kit de détection et procédé de détection de molécule cible Download PDF

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WO2024085226A1
WO2024085226A1 PCT/JP2023/037861 JP2023037861W WO2024085226A1 WO 2024085226 A1 WO2024085226 A1 WO 2024085226A1 JP 2023037861 W JP2023037861 W JP 2023037861W WO 2024085226 A1 WO2024085226 A1 WO 2024085226A1
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Prior art keywords
well
wells
target molecule
target
detecting
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PCT/JP2023/037861
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English (en)
Japanese (ja)
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陽介 堀内
涼輔 横田
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Ｔｏｐｐａｎホールディングス株式会社
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Publication of WO2024085226A1 publication Critical patent/WO2024085226A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N37/00—Details not covered by any other group of this subclass

Definitions

  • the present invention relates to a detection kit and a method for detecting a target molecule.
  • This application claims priority to Japanese Patent Application No. 2022-168341 filed in Japan on October 20, 2022, and Japanese Patent Application No. 2022-168342 filed on October 20, 2022, the contents of which are incorporated herein by reference.
  • Quantitative detection of target molecules in biological samples is used to detect diseases early and predict the effectiveness of medication.
  • the target molecule is a protein
  • quantification is performed using enzyme-linked immunosorbent assay (ELISA) or similar.
  • ELISA enzyme-linked immunosorbent assay
  • nucleic acid quantification is performed using real-time PCR or similar.
  • Patent Document 1 Patent Document 1, Patent Document 2, and Non-Patent Document 1 describe a technology for performing an enzyme reaction in multiple microcompartments to detect single molecules. These methods are called digital measurement.
  • the sample solution is filled into a reaction vessel (also called a detection device) that has an extremely large number of microcompartments, and divided into each of the microcompartments.
  • the signal from each microcompartment is then binarized, and the number of target molecules contained in the sample solution is measured by determining whether or not they are present in the microcompartment.
  • Digital measurement can significantly improve detection sensitivity and quantitation compared to conventional ELISA, real-time PCR, and other methods.
  • Digital measurement technology distributes target molecules contained in a solution sample one molecule at a time into microcompartments, and then counts the number of microcompartments into which the target molecules are distributed.
  • target molecules also called the target substance
  • conventional digital measurement technology is capable of detecting cells (i.e., the target substance) contained in a solution sample with high accuracy, it is difficult to detect and quantify cytokines (i.e., the target molecules) secreted from each cell with high accuracy.
  • the present invention has been made in consideration of these circumstances, and aims to provide a detection kit that enables highly sensitive detection of target molecules released from a target substance in solution. It also aims to provide a method for detecting target molecules using such a detection kit.
  • one aspect of the present invention includes the following aspects.
  • a detection kit comprising a detection device for target molecules and a sealing liquid used in the detection device, the sealing liquid being a mixture of a lipophilic liquid and a surfactant, the concentration of the surfactant in the sealing liquid being 1% by volume or more and 100% by volume or less with respect to the saturated concentration of the surfactant in the sealing liquid, the detection device comprising a first well plate having a plurality of first wells on one surface and a second well plate having a plurality of second wells on one surface, the first well plate and the second well plate being arranged such that the first wells and the second wells face each other, a flow path through which a fluid flows is formed between the first well plate and the second well plate, and the first wells overlap the second wells in a plan view.
  • a detection kit comprising a detection device for target molecules and a sealing liquid used in the detection device, the sealing liquid being a mixture of a lipophilic liquid and a surfactant, the concentration of the surfactant in the sealing liquid being 1% by volume or more and 100% by volume or less with respect to the saturated concentration of the surfactant in the sealing liquid, the detection device comprising a substrate, a wall material provided on the substrate, and a lid material facing the substrate and in contact with the wall material, the lid material having an inlet and an outlet penetrating in the thickness direction, a flow path through which a fluid flows formed between the top of the wall material and the lid material, and a plurality of first wells surrounded by the substrate and the wall material.
  • a method for detecting a target molecule using the detection kit described in [1-1], comprising the steps of filling each of the first wells with a sample solution containing a target substance and sealing the sample solution in the first wells by isolating them with the sealing liquid; releasing the target molecule from the target substance in one of the first wells in which the target substance is placed; filling the second wells with a buffer and pressing the surface of the second well in the second well plate against the surface of the first well in the first well plate to contact the surface of the second well in the second well plate, distributing the target molecule one molecule at a time to each of the second wells independent of the first well; and causing a reaction between the target molecule and a detection reagent to occur and detecting the target molecule.
  • one embodiment of the present invention includes the following:
  • a method for detecting a target molecule using a detection device comprising a first well plate having a plurality of first wells on one surface thereof and a second well plate having a plurality of second wells on one surface thereof, the first well plate and the second well plate being used with the first well and the second well facing each other, the method comprising the steps of: filling each of the plurality of first wells with a sample solution containing a target substance, and sealing the sample solution in the first well by using a sealing liquid to isolate the first well; obtaining a particulate evaluation object containing the target molecule from the target substance in the first well in which one of the target substances is disposed; filling the second well with a buffer, and contacting a surface of the first well in the first well plate with a surface of the second well in the second well plate, and distributing the evaluation object one by one to each of the second wells independent of the first well; and causing a reaction between the evaluation object and a detection reagent, and detecting the target
  • T d1 / v ... (1)
  • T (unit: sec) is the contact time.
  • d1 (unit: ⁇ m) is the diameter of the evaluation object.
  • v (unit: ⁇ m/sec) is the migration speed of the evaluation substance in the sample solution from the first well to the second well.
  • the present invention provides a detection kit that enables highly sensitive detection of target molecules released from a target substance in a solution. It also provides a method for detecting target molecules using such a detection kit.
  • FIG. 1 is a schematic diagram showing a detection kit 500 according to the first embodiment.
  • FIG. 2 is a cross-sectional view taken along line II-II in FIG.
  • FIG. 3 is a cross-sectional view showing another embodiment of the detection device 100.
  • FIG. 4 is an explanatory diagram of the method for detecting a target molecule according to the first embodiment.
  • FIG. 5 is an explanatory diagram of a method for detecting a target molecule according to the first embodiment.
  • FIG. 6 is an explanatory diagram of a method for detecting a target molecule according to the first embodiment.
  • FIG. 7 is an explanatory diagram of a method for detecting a target molecule according to the first embodiment.
  • FIG. 4 is an explanatory diagram of the method for detecting a target molecule according to the first embodiment.
  • FIG. 5 is an explanatory diagram of a method for detecting a target molecule according to the first embodiment.
  • FIG. 6 is an explanatory diagram of a method for
  • FIG. 8 is an explanatory diagram of a method for detecting a target molecule according to the first embodiment.
  • FIG. 9 is an explanatory diagram of a method for detecting a target molecule according to the first embodiment.
  • FIG. 10 is an explanatory diagram of the method for detecting a target molecule according to the first embodiment.
  • FIG. 11 is a cross-sectional view taken along the arrows and showing a detection device 200 of this embodiment.
  • FIG. 12 is an explanatory diagram of a method for detecting a target molecule according to the second embodiment.
  • FIG. 13 is an explanatory diagram of a method for detecting a target molecule according to the second embodiment.
  • FIG. 14 is an explanatory diagram of a method for detecting a target molecule according to the second embodiment.
  • FIG. 14 is an explanatory diagram of a method for detecting a target molecule according to the second embodiment.
  • FIG. 15 is a schematic perspective view showing a detection device 300 included in the detection kit according to the third embodiment.
  • 16 is a cross-sectional view taken along line XV-XV in FIG.
  • FIG. 17 is an explanatory diagram of a method for detecting a target molecule according to the third embodiment.
  • FIG. 18 is an explanatory diagram of a method for detecting a target molecule according to the third embodiment.
  • FIG. 19 is a schematic cross-sectional view showing a detection device 400 included in the detection kit according to the fourth embodiment.
  • FIG. 20 is an explanatory diagram of a method for detecting a target molecule according to the fourth embodiment.
  • FIG. 21 is an explanatory diagram of a method for detecting a target molecule according to the fourth embodiment.
  • FIG. 20 is an explanatory diagram of a method for detecting a target molecule according to the fourth embodiment.
  • FIG. 22 is an enlarged photograph showing the results of the example.
  • FIG. 23 is an enlarged photograph showing the results of the example.
  • FIG. 24 is an enlarged photograph showing the results of the example.
  • FIG. 25 is an enlarged photograph showing the results of the comparative example.
  • FIG. 26 is an enlarged photograph showing the results of the comparative example.
  • FIG. 27 is an enlarged photograph showing the results of the comparative example.
  • FIG. 28 is an enlarged photograph showing the results of an experimental example.
  • FIG. 29 is an enlarged photograph showing the results of an experimental example.
  • FIG. 30 is an enlarged photograph showing the results of an experimental example.
  • FIG. 31 is an enlarged photograph showing the results of an experimental example.
  • FIG. 32 is an enlarged photograph showing the results of an experimental example.
  • Detection kit 1 is a schematic diagram showing a detection kit 500 of the present embodiment.
  • the detection kit 500 includes a detection device 100 and an oily sealing liquid L2.
  • the sealing liquid L2 is stored in, for example, a storage container 50.
  • the detection kit 500 is used to detect a target molecule contained in a liquid sample.
  • the detection kit 500 may include a detection reagent for detecting the target molecule.
  • FIG. 2 is a cross-sectional view of the detection device 100 taken along line II-II in FIG.
  • the detection device 100 has a first well plate 11, a second well plate 12, and a wall material 13.
  • the detection device 100 is used as a reaction vessel that contains a sample therein, releases target molecules from a target substance contained in the sample, and performs a detection reaction of the target molecules.
  • the target molecules are released from the target substance contained in the sample in the first well plate 11, and the target molecules that migrate from the first well plate are detected in the second well plate 12.
  • the first well plate 11 and the second well plate 12 are stacked via a wall material 13 so that they can be easily attached and detached.
  • the first well plate 11 is a plate-like member having a rectangular shape in a plan view. "Plan view” refers to a field of view from the normal direction of the top surface of the first well plate 11.
  • first well plate 11 may be abbreviated as “first plate 11”.
  • second well plate 12 may be abbreviated as "second plate 12".
  • the first plate 11 has a plurality of first wells W1 on a well-forming surface 11a facing the inner surface of the detection device 100.
  • the first wells W1 are recesses provided in the well-forming surface 11a of the first plate 11 and open to the well-forming surface 11a.
  • the second plate 12 is a plate-like member that is rectangular in plan view and has the same contour shape as the first plate 11 in plan view.
  • the first plate 11 and the second plate 12 face the first well W1 and the second well W2, and are spaced apart from each other.
  • the second plate 12 has a plurality of second wells W2 on a well-forming surface 12a facing the inner surface of the detection device 100.
  • the second wells W2 are recesses provided in the well-forming surface 12a of the second plate 12 and open to the well-forming surface 12a.
  • the materials of the first plate 11 and the second well plate may or may not be electromagnetically transparent.
  • electromagnetic waves that are the subject of the transparency judgment include X-rays, ultraviolet rays, visible light, and infrared rays.
  • the first plate 11 and the second well plate are electromagnetically transparent, it becomes possible to use the electromagnetic waves to analyze the results of experiments performed with the detection device 100.
  • the fluorescence or phosphorescence that occurs as a result of irradiating electromagnetic waves can be measured via the first plate 11 or the second well plate.
  • electromagnetic transparency refers to the property of being able to transmit electromagnetic waves of various wavelengths, such as radio waves, light, X-rays, and gamma rays.
  • Examples of materials that are electromagnetically transparent include glass and resin.
  • resins include ABS resin, polycarbonate, COC (cycloolefin copolymer), COP (cycloolefin polymer), acrylic resin, polyvinyl chloride, polystyrene, polyethylene, polypropylene, polyvinyl acetate, PET (polyethylene terephthalate), PEN (polyethylene naphthalate), PC (polycarbonate), silicone resin, fluororesin, and amorphous fluororesin.
  • These resins may contain various additives, or may be polymer alloys in which multiple resins are mixed.
  • the electromagnetic wave transparent material preferably has substantially no autofluorescence. Having substantially no autofluorescence means that the material has no autofluorescence at the wavelength used for sample detection, or if it does have autofluorescence, it is so weak that it does not affect sample detection. For example, if the autofluorescence is about 1/2 or 1/10 of the fluorescence to be detected, it can be said to be so weak that it does not affect sample detection. If the first plate 11 and the second well plate are formed using such a material, the detection sensitivity can be increased in sample detection using electromagnetic waves.
  • An example of a material that is electromagnetically transparent and does not emit autofluorescence is quartz glass.
  • Materials that emit weak autofluorescence and do not interfere with sample detection using electromagnetic waves include low-fluorescence glass, acrylic resin, COC (cycloolefin copolymer), and COP (cycloolefin polymer), etc.
  • the material of the first plate 11 is preferably softer (i.e., has a lower modulus of elasticity) than the material of the second plate 12.
  • silicone resin is preferable as the material of the first plate 11.
  • PDMS polydimethylsiloxane
  • Both the material of the first plate 11 and the material of the second plate 12 may be PDMS.
  • the first plate 11 and the second plate 12 may be a single layer made of only the above material, or may be a laminate of multiple materials.
  • the layer having the well (first well W1 or second well W2) and the layer supporting said layer may be made of different materials.
  • a laminate in which a fluororesin is laminated on a substrate may be used as the material for the first plate 11, and the layer of fluororesin may be processed to form a well array.
  • CYTOP registered trademark
  • Asahi Glass or the like may be used as the fluororesin.
  • the thickness of the second plate 12 can be determined appropriately. When observing the fluorescence from the second plate 12 side using a fluorescent microscope, the thickness of the second plate 12 may be, for example, 5 mm or less, 2 mm or less, or 1.6 mm or less. The thickness of the first plate 11 can be determined appropriately as long as it does not impair the effects of the invention.
  • the first well W1 contains a sample contained inside the detection device 100, and is used as a site for releasing target molecules from a target substance contained in the sample.
  • the second well W2 receives the target molecules released in the first well W1 and functions as a reaction field between the target substance and a detection reagent for the target substance.
  • the first well W1 and the second well W2 may have various shapes.
  • cylindrical shapes such as a cylindrical shape, an elliptical cylindrical shape, and a polygonal cylindrical shape, pyramid shapes such as a cone shape and a pyramid shape, and truncated pyramid shapes such as a truncated cone shape and a truncated pyramid shape can be used.
  • pyramid shapes such as a cone shape and a pyramid shape
  • truncated pyramid shapes such as a truncated cone shape and a truncated pyramid shape
  • the opening diameter gradually decreases in the depth direction of the well.
  • the bottoms of the first well W1 and the second well W2 may be flat or curved (convex or concave).
  • the diameter of the opening of the first well W1 (also called the opening diameter) may be, for example, about 10 to 500 ⁇ m.
  • the diameter of the opening of the second well W2 (also called the opening diameter) may be, for example, about 1 to 100 ⁇ m. It is preferable that the opening diameter of the first well W1 is larger than the opening diameter of the second well W2.
  • the depth of the first well W1 may be, for example, about 5 to 500 ⁇ m.
  • the "depth of the first well W1" refers to the distance from the “imaginary plane that is parallel to and in contact with the well-forming surface 11a" to the "deepest part of the first well W1.”
  • the depth of the second well W2 may be, for example, about 1 to 100 ⁇ m.
  • the "depth of the second well W2" refers to the distance from the “imaginary plane that is parallel to and in contact with the well-forming surface 12a" to the "deepest part of the second well W2.”
  • the detection device 100 has 10 to 10,000 first wells W1.
  • the first wells W1 are arranged so as to overlap with the second wells W2 in a planar view.
  • the first wells W1 may be configured so as to overlap with the second wells W2 in a 1:1 ratio in a planar view, or may be configured so as to overlap with two or more second wells W2 in a planar view.
  • the number of second wells W2 that overlap one first well W1 in a planar view is 1 to 10,000, and preferably 10 to 10,000.
  • the second plate 12 should have a number of second wells W2 formed that satisfies the above number.
  • the first plate 11 and the second plate 12 described above can be manufactured using known injection molding, microimprinting technology, or nanoimprinting technology.
  • the first plate 11 and the second plate 12 can also be manufactured using known photolithography technology by forming wells by etching.
  • the wall material 13 is formed in a closed ring shape in a plan view, and is sandwiched between the first plate 11 and the second plate 12.
  • the wall material 13 functions as a spacer that separates the first plate 11 and the second plate 12 and creates a space between the first plate 11 and the second plate 12.
  • the material of the wall material 13 can be the same as that of the first plate 11 and the second plate 12 described above.
  • the wall material 13 made of such a material can be integrated with the first plate 11 or the second plate 12 by bonding with an adhesive or by welding with a method such as thermal welding, ultrasonic welding, or laser welding.
  • the material of the wall material 13 may be different from that of the first plate 11 and the second plate 12.
  • the wall material 13 may be, for example, a double-sided tape or an adhesive.
  • the base material of the double-sided tape may be plastic or paper.
  • the adhesive may be grease or a rapid-curing adhesive containing a component such as ⁇ -cyanoacrylate.
  • the wall material 13 may be formed simultaneously (i.e., integrally) with either the first plate 11 or the second plate 12 during the manufacturing process, and may be integral with either the first plate 11 or the second plate 12.
  • Figure 3 is a cross-sectional view showing another form of the detection device 100.
  • the first member 110 has a first plate 11, a first lid material 21 that is detachable from the first plate 11, and a first wall material 31 that is sandwiched between the first plate 11 and the first lid material 21.
  • the first lid material 21 has the same contour shape as the first plate 11 when viewed in a plan view.
  • the first lid material 21 has two through holes that penetrate in the thickness direction.
  • the two through holes are provided at one end side and the other end side of the first lid material 21.
  • One of the through holes is an inlet 211 used when injecting liquid into the internal space S1 of the first member 110, and the other through hole is an outlet 212 used when discharging liquid from the internal space S1.
  • liquid includes not only liquid samples, but also detection reagents and sealing liquids.
  • the inlet 211, the internal space S1, and the outlet 212 are connected in this order, and together they form a flow path FC1.
  • a liquid material is appropriately caused to flow through the flow path FC1, and the target substance is sealed in the first well W1.
  • multiple first wells W1 are arranged between the inlet 211 and the outlet 212.
  • a cylindrical injection port 215 is provided on the upper surface 21a of the first lid member 21, surrounding the injection port 211.
  • the injection port 215 is connected to the injection port 211.
  • the injection port 215 is used to connect a syringe, for example, when filling the internal space with a liquid using a syringe filled with the liquid.
  • a cylindrical discharge port 216 is provided on the upper surface 21a of the first lid member 21, surrounding the periphery of the discharge outlet 212.
  • the discharge port 216 is connected to the discharge outlet 212.
  • the discharge port 216 is used, for example, to connect a tube through which the liquid flows when the liquid is extracted from the internal space S1.
  • the second member 120 also has a second plate 12, a second lid material 22 that is detachable from the second plate 12, and a second wall material 32 that is sandwiched between the second plate 12 and the second lid material 22.
  • the second lid material 22 has the same contour shape as the second plate 12 when viewed in a plan view.
  • the second lid member 22 has an inlet 221 and an outlet 222 that penetrate in the thickness direction.
  • the inlet 221, the internal space S2 of the second member 120, and the outlet 222 are connected in this order, and as a whole form a flow path FC2.
  • a liquid buffer is caused to flow through the flow path FC2 and sealed in the second well W2.
  • multiple second wells W2 are arranged between the inlet 221 and the outlet 222.
  • the upper surface 22a of the second lid member 22 is provided with a cylindrical injection port 225 that surrounds the injection port 221 and communicates with the injection port 221.
  • the upper surface 22a is provided with a cylindrical exhaust port 226 that surrounds the exhaust port 212 and communicates with the exhaust port 212.
  • the materials for the first lid material 21 and the second lid material 22 can be the materials exemplified above as the materials for the first plate 11 and the second plate 12.
  • the first lid material 21 and the second lid material 22 can be manufactured by known injection molding.
  • the sealing liquid L2 is a mixture of a lipophilic liquid and a surfactant.
  • a lipophilic liquid so-called oil
  • a fluorine-based oil a silicone-based oil, a hydrocarbon-based oil, or a mixture thereof can be used.
  • the lipophilic liquid may be Sigma's product name "FC-40" (CAS number: 86508-42-1).
  • FC-40 is a fluorinated aliphatic compound (fluorine-based oil) with a specific gravity of 1.85 g/mL at 25°C.
  • the surfactant either an ionic surfactant or a non-ionic surfactant can be used. Furthermore, it is preferable to use a surfactant that can generally be used in biochemical experiments. When cells are used as the target substance, it is preferable to use a surfactant that does not destroy cell membranes or that does not destroy cell membranes easily. When cells are used as the target substance, it is advisable to adjust the concentration of the surfactant in the sealing solution to a concentration that does not cause cell alteration or destruction.
  • Ionic surfactants include anionic surfactants and amphoteric surfactants.
  • anionic surfactant include sodium dodecyl sulfate, sodium cholate, and sodium deoxycholate.
  • Amphoteric detergents include, for example, CHAPS and CHAPSO.
  • CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate
  • CHAPSO 3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxypropanesulfonate
  • Triton X-100 has 9 or 10 repeating polyethylene glycol moieties, while Triton X-114 has 7 or 8 repeating polyethylene glycol moieties.
  • the concentration of the surfactant in the sealing liquid L2 is preferably 1% by volume or more and 100% by volume or less, and more preferably 10% by volume or more and 90% by volume or less, relative to the saturated concentration of the surfactant in the sealing liquid.
  • the appropriate surfactant concentration differs depending on the type of lipophilic liquid used in the sealing liquid L2. Therefore, it is advisable to adjust the surfactant concentration by conducting preliminary experiments as appropriate.
  • the sealing liquid L2 may be diluted before use in the detection method described below. Therefore, the detection kit 500 may further include only a lipophilic solution that does not contain a surfactant, which may be used to dilute the sealing liquid L2.
  • Method 1 for detecting target molecules 4 to 9 are explanatory diagrams of the method for detecting a target molecule according to the present embodiment.
  • the method for detecting a target molecule includes the steps of carrying out the following operations.
  • the above-mentioned detection device 100 is used to detect a target molecule contained in a sample in the second well W2 of the detection device 100.
  • a step of adjusting the concentration of a liquid sample containing a target substance to obtain a sample solution A step of sealing the sample solution in a first well independently using a sealing liquid;
  • a step of releasing a target molecule from the target substance in the first well A step of distributing one target molecule per second well;
  • A5 A step of detecting the target molecule.
  • step (A1) Step of adjusting the concentration of a liquid sample containing a target substance to obtain a sample solution
  • the concentration of a liquid sample containing a target substance is adjusted to obtain a sample solution L1.
  • this step may be abbreviated as "step (A1)".
  • each step may be abbreviated in the same manner.
  • Samples include, for example, biological samples or environmental samples.
  • Biological samples are not particularly limited, and examples include serum, plasma, urine, and cell culture fluid.
  • Environmental samples include, for example, river water and industrial wastewater.
  • Biological samples and environmental samples contain target substances that release target molecules to be detected.
  • target substances include DNA, RNA, proteins, viruses, cells, and specific compounds.
  • RNA include miRNA and mRNA.
  • cells include bacteria, yeast, animal cells, plant cells, and insect cells.
  • the sample may contain a reaction reagent for detecting the target substance.
  • the reaction reagent include a buffer substance, an enzyme, a substrate, an antibody, and an antibody fragment.
  • the enzyme is selected according to the content of the biochemical reaction in order to carry out a biochemical reaction such as an enzyme reaction on a template nucleic acid related to the target substance.
  • the biochemical reaction on the template nucleic acid is, for example, a reaction in which signal amplification occurs under conditions in which the template nucleic acid is present.
  • an enzyme may be bound to the antibody and detected by an enzyme reaction.
  • the detection enzyme may be HRP, alkaline phosphatase, ⁇ -galactosidase, or the like.
  • the reaction reagent is selected according to the detection reaction to be adopted.
  • Specific detection reactions include the Invasive Cleavage Assay (ICA) method, the Loop-Mediated Isothermal Amplification (LAMP) method (registered trademark), the 5' ⁇ 3' nuclease method (TaqMan (registered trademark) method), and the fluorescent probe method.
  • ICA Invasive Cleavage Assay
  • LAMP Loop-Mediated Isothermal Amplification
  • TaqMan registered trademark
  • fluorescent probe method the fluorescent probe method.
  • the sample may contain a surfactant.
  • the concentration of the sample is adjusted based on the volume of the detection device 100 used so that one target substance M1 is placed in each first well W1.
  • the concentration of the sample should be determined by performing a preliminary experiment using the detection device 100.
  • step (A2) Step of sealing the sample solution in the first wells by sealing liquid
  • the sample solution L1 is filled into each of the first wells W1 independently.
  • this step may be abbreviated as “step (A2)".
  • the detection device 100 is divided into a first member 110 and a second member 120, and the first member 110 is used in this step.
  • a sample solution L1 containing a detection reagent is injected into the internal space S1 from the injection port 211.
  • the sample solution L1 flows through the internal space S1 (also called the flow path FC1), the sample solution L1 fills the first well W1 that opens into the internal space S1.
  • the sample solution L1 that has passed through the flow path FC1 is discharged from the outlet 212.
  • one target substance M1 is filled into one first well W1.
  • the concentration of the sample solution L1 is adjusted so that the number of target substances M1 injected into the internal space S1 is, for example, 1/10 or less of the total number of first wells W1.
  • one first well W1 is filled with one or less target substances M1, i.e., 0 or 1 target substance M1.
  • the means for introducing the target substance M1 into the first well W1 is not particularly limited, and an appropriate means can be selected according to the target substance M1 to be detected.
  • the target substance M1 may be distributed into the first well W1 by its own weight, or may be introduced into the first well W1 by distribution due to diffusion.
  • a substance that captures the target substance M1 (also called a capture material) may be used, and the capture material may be bound to the target substance M1 that does not easily settle under its own weight and then delivered. Furthermore, the efficiency of introducing the target substance M1 into the first well W1 can be improved by fixing the capture material to the first well W1 in advance and allowing the capture material to capture the target substance M1 delivered together with the sample solution.
  • the target substance M1 may be introduced into the first well W1, and the capture object and target substance M1 may be brought into contact in the first well W1.
  • the capture substance is a substance that can capture the target substance M1.
  • the capture substance may be, for example, a conjugate between a solid phase and a substance that specifically binds to the target substance M1.
  • Antibody fragments include Fab, F(ab')2, Fab', single-chain antibodies (scFv), disulfide-stabilized antibodies (dsFv), dimeric V-domain fragments (diabodies), and peptides containing CDRs.
  • the antibodies may be monoclonal or polyclonal. They may also be commercially available antibodies.
  • the specific binding substance when the target substance M1 includes a glycan, the specific binding substance may be a lectin. In addition, when the target substance M1 includes a lipid membrane, the specific binding substance may be a substance that has binding properties to lipid membranes. Examples of substances that have binding properties to lipid membranes include hydrocarbons such as hexanediol and membrane proteins such as transmembrane proteins. Examples of membrane proteins include ⁇ -hemolysin.
  • the solid phase constituting the complement may be a particle, a membrane, a substrate, or the like.
  • the substance that specifically binds to the target substance M1 may be one type, or two or more types. For example, there may be three types, four types, or five or more types.
  • the particles are not particularly limited, and examples thereof include polymer particles, magnetic particles, and glass particles.
  • the particles are preferably surface-treated to avoid non-specific adsorption.
  • particles having functional groups such as carboxyl groups on the surface are preferable to immobilize specific binding substances. More specifically, products such as "Magnosphere LC300" manufactured by JSR Corporation can be used.
  • the method for immobilizing a specific binding substance on a solid phase is not particularly limited, and examples include physical adsorption, chemical binding, avidin-biotin binding, and binding between protein G or protein A and an antibody.
  • Physical adsorption methods include methods in which specific binding substances are fixed to the particle surface by hydrophobic or electrostatic interactions.
  • Chemical bonding methods include those that use crosslinking agents.
  • the specific binding substance can be immobilized on the particle surface by reacting the carboxyl groups of the specific binding substance with a crosslinking agent to form an active ester, and then reacting the hydroxyl groups with the ester groups.
  • cells to which the virus can attach i.e., cells having a virus receptor
  • the capture substance may be used as the capture substance.
  • sealing liquid L2 is injected into the internal space S1 through the injection port 211.
  • the sealing liquid L2 may be the original sealing liquid L2 contained in the storage container 50, or may be a diluted liquid of the sealing liquid L2.
  • the sealing liquid L2 flows in the internal space S1 in the direction of the well formation surface 11a, and displaces the internal space S1 by washing away the sample solution L1 that is not contained in the first well W1 among the sample solution L1 sent to the flow path FC1.
  • the sealing liquid L2 individually seals each of the multiple first wells W1, and the first wells W1 containing the sample solution L1 become independent reaction spaces.
  • the sample solution L1 replaced with the sealing liquid L2 and the excess sealing liquid L2 are discharged from the discharge port 212.
  • step (A3) Step of Releasing Target Molecules from the Target Substance in the First Well
  • the target molecules are released from the target substance M1 distributed to each first well W1.
  • this step may be abbreviated as “step (A3)”.
  • the method for releasing the target molecule from the target substance is selected depending on the type of target substance. For example, if the target substance is DNA or RNA, multiple fragments (i.e., target molecules) may be obtained from one fragment by ultrasonic disruption or enzymatic degradation.
  • the detection device 100 may be heated to separate the individual nucleic acids through thermal denaturation.
  • the target substance is a protein aggregate
  • the aggregates may be loosened by ultrasonic irradiation, an enzyme that breaks down proteins may be used, or a reagent that breaks down protein aggregates may be added.
  • the cell may be disrupted by ultrasound or the cell membrane may be disrupted by applying electrical stimulation.
  • Specific methods include the following: First, beads are placed in the first well W1.
  • the beads may be placed in the first well W1 in advance, or may be added to a solution containing cells and placed in the first well W1 together with the cells.
  • There are no limitations on the size of the beads as long as they do not impair the effects of the invention and are large enough to be placed in the first well W1.
  • the material of the beads may be silica or a magnetic material.
  • the size of the beads is preferably 50 nm to 500 ⁇ m, more preferably 100 nm to 100 ⁇ m, and even more preferably 500 nm to 10 ⁇ m.
  • the first well W1 is isolated by the above-mentioned step (A2) and the cells are cultured. After culturing the cells for a certain period of time, ultrasonic waves are irradiated to the detection device 100. By vibrating the beads in the first well W1, the cell walls of the cells are broken down by the beads, and the contents of the cells can be released into the first well W1.
  • the cell membrane may be dissolved using a reagent that dissolves the cell membrane.
  • a solution containing cells is mixed with a reagent that dissolves cell membranes (also called a cell lysis solution).
  • the solution containing cells and the cell lysis solution may be mixed before being injected into the detection device 100, or may be mixed within the detection device 100.
  • one method is to first inject the solution containing cells into the first well W1, and then inject the cell lysis solution into the first well W1.
  • Possible methods for adding the cell lysis solution to the first well W1 include adding it to the detection device 100 at a flow rate that does not cause the cells placed in the first well W1 to leave the first well W1, or fixing the cells in the first well W1.
  • the method of fixing the cells in the first well W1 may be a method of binding a cell capture substance to the first well W1, or a method of placing particles with a capture substance bound to them in the first well W1.
  • An example of a "capture substance” is an antibody that binds to cells.
  • surfactants or commercially available reagents can be used.
  • the first well W1 is separated in step (A2) and the cell membrane is dissolved by reacting for a certain period of time. This allows the contents of the cells to be released into the first well W1.
  • cells which are the target substance, may be cultured for a certain period of time in the first well W1, and secretions such as cytokines secreted by the cells may be detected as target molecules.
  • step (A4) Step of distributing one target molecule to each second well
  • the target molecule released into the sample solution L1 is distributed one molecule to each second well W2.
  • this step may be abbreviated as “step (A4)”.
  • liquid buffer B is injected into the internal space S2 from the injection port 221 of the second member 120.
  • Buffer B may contain a detection reagent for the target molecule.
  • buffer B flows through the internal space S2 (flow path FC2), buffer B fills the second well W2 that opens into the internal space S2.
  • the buffer B that has passed through flow path FC2 is discharged from outlet 222.
  • sealing liquid L2 is injected into the internal space S2 from the injection port 221.
  • the sealing liquid L2 flows in the internal space S1 in the planar direction of the well formation surface 12a, and displaces the buffer B that is not contained in the second well W2 out of the buffer B sent to the flow path FC2, thereby replacing the internal space S2.
  • the sealing liquid L2 may be the same as the sealing liquid used in step (A2) above.
  • the sealing liquid used in step (A2) and the sealing liquid used in this step may be the same or different.
  • the sealing liquid L2 individually seals each of the multiple second wells W2, and the second wells W2 containing buffer B become independent.
  • the buffer B replaced by the sealing liquid L2 and the excess sealing liquid L2 are discharged from the discharge port 222.
  • first lid material 21 is removed from the first member 110 to form the first plate 11.
  • second lid material 22 is removed from the second member 120 to form the second plate 12.
  • the well formation surfaces 11a and 12a of these well plates are covered with sealing liquid L2.
  • the well-forming surface 11a of the first plate 11 and the well-forming surface 12a of the second plate 12 are placed opposite each other to form the detection device 100.
  • the first well W1 is positioned so as to overlap the second well W2 in a plan view.
  • the first plate 11 and the second plate 12 are pressed against each other with pressure, and the well-forming surface 11a and the well-forming surface 12a are brought into contact.
  • the experimenter may press them directly with their hands, or may use a tool such as a clip or roller.
  • the pressure pressing the first plate 11 and the second plate 12 together may be the experimenter's own strength, or may be applied using a jig such as a weight or clip.
  • the first well W1 and the second well W2 are connected, and the target molecules M2 in the first well W1 diffuse and move to the second well W2.
  • the sealing liquid containing a surfactant effectively facilitates the transfer of material from the first well W1 to the second well W2 in this process.
  • the sealing liquid L2 contains a surfactant
  • the surface tension of the sealing liquid L2 is smaller than when the sealing liquid L2 does not contain a surfactant, and it is believed that the sealing liquid L2 between the first well W1 and the second well W2 is more likely to penetrate.
  • This makes it easier to form a state in which the first well W1 and the second well W2 are connected as shown in FIG. 9, and it is believed that the transfer of material from the first well W1 to the second well W2 is more effective.
  • the target molecule M2 that has migrated to the second well W2 may be dissolved in the sample solution L1 in the second well W2, or may be captured by a target molecule capture substance fixed to the second well W2.
  • the target molecule capture substance is a substance that has a functional group that specifically binds to the target molecule M2, and may be, for example, an antibody if the target molecule M2 is a protein, or a nucleic acid that forms a complementary strand if the target molecule M2 is a nucleic acid.
  • target molecules M2 may be released from the target substance M1 in the first well W1, and the released target molecules M2 may be captured by the capture particles.
  • the capture particles that have captured the target molecules M2 may be moved from the first well W1 to the second well W2, thereby introducing the target molecules M2 into the second well W2.
  • the difference in specific gravity between the sample solution L1 and the capture particles may be utilized to cause the capture particles to settle or float, and the capture particles may be moved from the first well W1 to the second well W2.
  • the behavior of the capture particles may be controlled using magnetic force from outside the detection device 100.
  • step (A5) Step of detecting a target molecule
  • the target molecule M2 is reacted with the detection reagent to detect the target molecule M2.
  • this step may be abbreviated as “step (A5)”.
  • One example of a detection method is to carry out a signal amplification reaction inside the sealed second well W2. Inside the second well W2, the signal derived from the detection reagent is amplified so that it can be detected.
  • Signals include fluorescence, color development, changes in potential, and changes in pH.
  • the signal amplification reaction may be, for example, a biochemical reaction, more specifically, an enzyme reaction.
  • the signal amplification reaction is an isothermal reaction in which a reagent solution containing an enzyme for signal amplification is contained in the second well W2, and the detection device 100 is maintained under constant temperature conditions at which the desired enzyme activity is obtained, for example, at least 60°C, preferably about 66°C, for a predetermined time, for example, at least 10 minutes, preferably about 15 minutes.
  • signal amplification reactions when the target molecule M2 is a nucleic acid, include ICA reactions such as the Invader (registered trademark) method, loop-mediated isothermal amplification (LAMP (registered trademark) method), 5' ⁇ 3' nuclease method (TaqMan (registered trademark) method), and fluorescent probe method. It is believed that these methods can cause a signal amplification reaction even if the solution contains the above-mentioned surfactant (cell lysis solution).
  • Invader registered trademark
  • LAMP loop-mediated isothermal amplification
  • TaqMan registered trademark
  • the solution in the second well W2 may be replaced.
  • the target molecule M2 may be immobilized in the second well W2 and then a detection reagent may be added from the outside.
  • the target molecule may be fixed in the second well W2 by binding a substance that captures the target molecule M2 to the second well W2, or by adding magnetic particles bound to a compound that captures the target molecule M2 to the second well W2 and fixing the magnetic particles by magnetic force.
  • an ICA reaction As a signal amplification reaction, it is particularly preferable to use an ICA reaction.
  • signal amplification proceeds through a cycle of two reactions: (1) complementary binding between nucleic acids, and (2) recognition and cleavage of a triple-stranded structure by an enzyme.
  • the effect of reaction cycle inhibition by contaminants other than the target molecule is small. Therefore, even if various components (contaminants) other than the target molecule are present in the second well W2, the target molecule can be detected with high accuracy by using an ICA reaction.
  • the sample solution L1 contains the reaction reagents and template nucleic acid necessary for the ICA reaction.
  • the biochemical reaction in the reaction step is an ICA reaction
  • the fluorescent substance is released from the quenching substance, thereby emitting a predetermined fluorescent signal in response to the excitation light.
  • Target molecules can also be detected by binding a substance that specifically binds to the target molecule (specific binding substance) to the target molecule and detecting the bound specific binding substance.
  • a substance that specifically binds to the target molecule specifically binding substance
  • the target molecule is a protein
  • it can be detected using ELISA. More specifically, detection can be performed by, for example, sandwich ELISA using the principle of FRET.
  • a first specific binding substance e.g., an antibody
  • a second specific binding substance labeled with a second fluorescent substance i.e., an acceptor
  • a target molecule e.g., an antigen
  • a target molecule e.g., an antigen
  • the distance between the donor and acceptor decreases, and the fluorescence wavelength of the acceptor can be detected by irradiation with the excitation wavelength of the donor.
  • the specific binding substance can be labeled with a nucleic acid fragment, and the nucleic acid fragment can be detected by an ICA reaction.
  • Specific binding substances can be the same as the specific binding molecules for the structures described below, such as antibodies, antibody fragments, and aptamers.
  • the specific binding substance can be directly or indirectly labeled with an enzyme such as horseradish peroxidase (HRP).
  • HRP horseradish peroxidase
  • a known appropriate method can be selected for observing the signal depending on the type of signal to be observed. For example, when performing bright-field observation, white light is irradiated perpendicularly onto the substrate on which the well array is provided. When observing fluorescent signals, excitation light corresponding to the fluorescent substance is irradiated into the well from the bottom side of the well, and the fluorescence emitted by the fluorescent substance is observed. An image of the entire or part of the observed well array is photographed and saved, and the image is processed by a computer system.
  • the detection device 100 configured as described above makes it possible to detect target molecules released from a target substance in a solution with high sensitivity.
  • the above-mentioned method for detecting a target molecule it is possible to analyze a target molecule released from a single target substance. Furthermore, it is possible to analyze the characteristics of the target substance that released the target molecule after detecting the target molecule, for example by analyzing the DNA or RNA of the cell after detecting a cytokine, or by counting the number of specific DNA sequences and then analyzing the entire sequence of that DNA. This makes it possible to detect the target molecule with high accuracy.
  • step (A1) is performed, but step (A1) may be omitted. If step (A1) is not performed, after filling the first wells with the sample solution, a first well W1 in which one target substance M1 is placed may be extracted from among the multiple first wells in step (A3), and the subsequent steps (A4) and (A5) may be performed.
  • [Second embodiment] 10 to 13 are explanatory diagrams of the detection device and the target molecule detection method of the detection kit according to the second embodiment.
  • the same components as those in the first embodiment are denoted by the same reference numerals, and detailed descriptions thereof will be omitted.
  • FIG. 10 is a cross-sectional view taken along an arrow showing a detection device 200 included in the detection kit of this embodiment, and corresponds to FIG.
  • the detection device 200 has a first well plate 15, a second plate 12, and a wall material 13.
  • the detection device 200 is used as a reaction vessel that contains a sample, releases target molecules from a target substance contained in the sample, and performs a detection reaction of the target molecules. In this case, the target molecules are released from the target substance contained in the sample in the first well plate 15, and the target molecules that migrate from the first well plate 15 are detected in the second plate 12.
  • the first well plate 15 and the second plate 12 are stacked via a wall material 13 so that they can be easily attached and detached.
  • the first well plate 15 is a plate-like member having a rectangular shape in a plan view. In the following description, the first well plate 15 may be abbreviated to "first plate 15.”
  • the first plate 15 has a plurality of first wells W1 on a well-forming surface 15a facing the inner surface of the detection device 200.
  • the first wells W1 are recesses provided in the well-forming surface 15a of the first plate 11 and open to the well-forming surface 15a.
  • the first plate 15 has two through holes that penetrate in the thickness direction.
  • the two through holes are provided at one end and the other end of the first plate 15, respectively.
  • One of the through holes is an inlet 151 used to inject liquid into the internal space S of the detection device 200, and the other through hole is an outlet 152 used to discharge liquid from the internal space S.
  • the inlet 151, the internal space S, and the outlet 152 are connected in this order, and together they form a flow path FC.
  • a liquid is appropriately caused to flow through the flow path FC, and the target substance is sealed in the first well W1.
  • the first plate 15 has a plurality of first wells W1 between the inlet 151 and the outlet 152 in a plan view.
  • the upper surface of the first plate 15 may have a cylindrical injection port surrounding the periphery of the inlet 151. Also, the upper surface of the first plate 15 may have a cylindrical injection port surrounding the periphery of the outlet 152.
  • [Method for detecting target molecules] 11 to 13 are explanatory diagrams of the method for detecting a target molecule of this embodiment.
  • the above-mentioned steps (A1) to (A5) are performed using a detection device 200.
  • Step (A1) is common to the first embodiment.
  • a sample solution L1 containing a detection reagent is injected from the injection port 151 into the internal space S (i.e., the flow path FC), and the first well W1 that opens into the internal space S is filled with the sample solution L1.
  • the second well W2 is also filled with the sample solution L1.
  • a sealing liquid L2 containing a surfactant is injected into the internal space S (i.e., the flow path FC) from the injection port 151.
  • the sealing liquid L2 flows in the internal space S in the planar direction of the well formation surface 11a, and washes away the sample solution L1 that is not contained in the first well W1 and the second well W2 in the internal space S, replacing the internal space S.
  • the sealing liquid L2 individually seals each of the multiple first wells W1 and second wells W2 (step (A2)).
  • the sample solution L1 replaced with the sealing liquid L2 and the excess sealing liquid L2 are discharged from the discharge port 152.
  • target molecules are released from the target substance M1 distributed to each first well W1 according to the method described above (step (A3)).
  • the detection device 200 is pressed against both sides of the first plate 15 and the second plate 12, bringing the well-forming surface 15a into contact with the well-forming surface 12a.
  • the first well W1 and the second well W2 are connected, and the target molecules M2 in the first well W1 diffuse and move to the second well W2 (step (A4)).
  • step (A5) a reaction between the target molecule M2 and the detection reagent is caused in the second well W2, and the target molecule M2 is detected.
  • the detection device 200 configured as described above makes it possible to detect target molecules released from a target substance in a solution with high sensitivity.
  • the above-described method for detecting target molecules also makes it possible to detect target molecules with high accuracy.
  • the detection device 200 of this embodiment has an inlet 151 and an outlet 152 in the first plate 15, this is not limited to this.
  • the detection device may have a first plate 11 and a second well plate 16 having an inlet 161 and an outlet 162.
  • the detection device 250 can be used in the same manner as the detection device 200 to carry out a method for detecting a target molecule.
  • FIG. 14 to 17 are explanatory diagrams of the detection device and the target molecule detection method of the detection kit according to the third embodiment.
  • the same components as those in the first embodiment are denoted by the same reference numerals, and detailed descriptions thereof will be omitted.
  • FIG. 14 is a schematic perspective view showing the fluidic device of this embodiment, and Fig. 15 is a cross-sectional view taken along line XV-XV in Fig. 14.
  • the fluidic device 300 has a well plate (also called a substrate) 17, a cover material 23, and a wall material 33.
  • the fluidic device 300 has a plurality of first wells W1 surrounded by the well plate 17 and the wall material 33, and a plurality of second wells W2 each provided on the top surface 17a of the well plate 17 at the bottom of the first wells W1.
  • the well plate 17 is a plate-like member having a rectangular shape in a plan view.
  • a plurality of second wells W2 are provided on an upper surface 17a of the well plate 17.
  • the material of the well plate 17 can be the same as that of the first well plate 11 described above.
  • the wall material 33 is formed in a closed ring shape in a plan view, and is disposed on the upper surface 17a of the well plate 17.
  • the wall material 33 is sandwiched between the well plate 17 and the cover material 23, and is integrated with them to form the fluidic device 300.
  • the space surrounded by the well plate 17, the cover material 23, and the wall material 33 is an internal space S in which a liquid sample is accommodated.
  • the wall material 33 functions as a wall surface of the internal space S, and also functions as a spacer between the well plate 17 and the lid material 23.
  • the internal space S is partitioned by wall material 33, and multiple first wells W1 are formed.
  • the first wells W1 refer to the space surrounded by the well plate 17, the wall material 33, and an imaginary plane that is parallel to and in contact with the top 33a of the wall material 33.
  • Multiple second wells W2 are arranged at the bottom of the first well W1.
  • the material of the wall material 33 can be the same as that of the well plate 17 described above.
  • the wall material 33 made of such a material can be integrated with the well plate 17 and the lid material 23 by bonding with an adhesive or by welding using heat welding, ultrasonic welding, laser welding, or the like.
  • the wall material 33 may be formed integrally with the well plate 17 and may constitute a part of the well plate 17. Similarly, the wall material 33 may be formed integrally with the lid material 23 and may constitute a part of the lid material 23.
  • the lid member 23 has the same contour shape as the well plate 17 in a plan view.
  • a convex portion 239 is provided on the outer edge of the lower surface 23b of the lid member 23.
  • the lid member 23 is connected to the wall material 33 via the convex portion 239.
  • the lid material 23 has two through holes (inlet 231, outlet 232) that penetrate in the thickness direction.
  • the top surface 23a of the lid material 23 is provided with a cylindrical injection port 235 that surrounds the periphery of the injection port 231.
  • the top surface 23a of the lid material 23 is provided with a cylindrical exhaust port 236 that surrounds the periphery of the exhaust port 232.
  • the material for the lid 23 can be the same as that for the first plate 11 described above.
  • a silicone resin is preferable as the material for the lid 23.
  • PDMS can be preferably used as the silicone resin.
  • the inlet 231, the internal space S, and the outlet 232 are connected in this order, and together they form a flow path FC.
  • a liquid is appropriately caused to flow through the flow path FC, and a detection reaction for the target substance is carried out.
  • Method for detecting target molecules 16 and 17 are explanatory diagrams of the method for detecting a target molecule according to this embodiment.
  • the method for detecting a target molecule is carried out using a detection kit including the above-mentioned detection device 300, and includes the steps of carrying out the following operations.
  • (B1) a step of sealing each second well by separating the buffer with a sealing liquid; (B2) a step of filling each of a plurality of first wells with a sample solution containing a target substance and sealing the sample solution in each of the first wells; (B3) a step of releasing a target molecule from the target substance in the first well; (B4) a step of distributing one target molecule per second well; (B5) a step of detecting the target molecule.
  • Step B1 Step of sealing each second well by isolating the buffer with a sealing liquid
  • the second well W2 of the detection device 300 is filled with buffer B, and each second well W2 is sealed by isolating it with a sealing liquid L2 containing a surfactant.
  • (B2) A step of filling each of the first wells with a sample solution containing a target substance and sealing the sample solution separately in the first well. Next, the concentration of the liquid sample containing the target substance is adjusted to obtain a sample solution L1.
  • the sample solution L1 can be prepared in the same manner as in the above-mentioned step (A1). At this time, the sample solution L1 does not contain a surfactant.
  • the prepared sample solution L1 is filled into each of the first wells W1.
  • sample solution L1 is injected into the internal space S from the injection port 231.
  • flow path FC the sample solution L1 pushes out the buffer B in the internal space S, and the first well W1 that opens into the internal space S is filled with the sample solution L1.
  • the sample solution L1 that has passed through the flow path FC is discharged from the outlet 232.
  • the internal space S is filled with the sample solution L1, and then the sealing liquid L21 is sent from the inlet 231 to the flow path FC.
  • the sealing liquid L21 any of the sealing liquids described above can be used.
  • the viscosity of the sealing liquid L21 is lower than the viscosity of the sealing liquid L2.
  • the sealing liquid L21 sent to the flow path FC flows over the first well W1 in the internal space S without entering the first well W1, and displaces the sample solution L1 that is not contained in the first well W1 among the sample solution L1 filled in the internal space S.
  • the sealing liquid L21 seals each of the multiple first wells W1 individually, and the first wells W1 containing the sample solution L1 become independent reaction spaces.
  • the target substance contained in the sample solution L1 is filled independently into each of the first wells W1.
  • the sample solution L1 replaced by the sealing liquid L21 and the excess sealing liquid L21 are discharged from the discharge port 232.
  • the first well W1 may be sealed using only the lipophilic liquid that constitutes the sealing liquid L2.
  • step (B3) Step of Releasing Target Molecules from the Target Substance in the First Well
  • the target molecules M2 are released from the target substance M1 distributed to each of the first wells W1.
  • step (B5) Step of Detecting Target Molecule
  • a reaction between the target molecule M2 and the detection reagent is caused to occur in the second well W2, and the target molecule M2 is detected.
  • a detection kit having a detection device 300 configured as described above can detect target molecules released from a target substance in a solution with high sensitivity.
  • the above-described method for detecting target molecules also makes it possible to detect target molecules with high accuracy.
  • [Fourth embodiment] 18 to 20 are explanatory diagrams of a method for detecting a target molecule according to the fourth embodiment.
  • the method for detecting a target molecule is carried out using a detection kit including a detection device 400, and includes the steps of carrying out the following operations.
  • (C1) A step of preparing a W/O type emulsion in which droplets containing a target substance are dispersed in a lipophilic liquid;
  • (C2) A step of sealing each well by isolating a buffer with a liquid containing a lipophilic liquid;
  • (C3) A step of flowing the emulsion through a flow path and replacing the liquid containing a lipophilic liquid with the emulsion;
  • (C4) A step of releasing target molecules from the target substance in the droplets;
  • (C5) A step of pressurizing the emulsion via a lid material and distributing droplets from the emulsion to each of the first wells independent of each other;
  • (C6) A step of detecting target molecules.
  • FIG. 18 is a schematic cross-sectional view showing a detection device 400.
  • the detection device 400 is a member having the same configuration as the second member 120 shown in the first embodiment, and has a well plate 12, a lid material 22 facing the well plate 12, and a wall material 32 sandwiched between the well plate 12 and the lid material 22.
  • the well plate 12, the lid material 22, and the wall material 32 have the same configuration as the respective members of the second member 120 shown in the first embodiment.
  • the same reference numerals as the second member 120 are used.
  • Well W corresponds to the first well in the present invention.
  • the well plate 12 is a member having the same configuration as the second member 12 described above.
  • the well plate 12 has a plurality of wells W (i.e., first wells).
  • the lid material 22 is the same as the second lid material 22 described above
  • the wall material 32 is the same as the second wall material 32 described above.
  • a W/O type emulsion in which droplets containing a target substance are dispersed in a lipophilic liquid is prepared using a target substance and a sealing liquid (i.e., a mixture of a surfactant and a lipophilic liquid).
  • a sealing liquid i.e., a mixture of a surfactant and a lipophilic liquid.
  • the concentration of the target substance, the concentration of the surfactant, or the amount of surfactant relative to the target substance is adjusted so that each droplet in the emulsion contains 0 or 1 target substance.
  • the target substance is distributed to each droplet in the emulsion.
  • the liquid L3 used for sealing may be the same as the dispersion medium of the emulsion, or it may be different.
  • the dispersion medium of the emulsion is the lipophilic liquid of the sealing liquid used in preparing the emulsion.
  • a fluorine-based oil can be used as the dispersion medium of the emulsion
  • a silicone-based oil can be used as the liquid L3 used for sealing.
  • liquid L3 may or may not contain a surfactant, as long as this does not impair the effects of the invention. If liquid L3 contains a surfactant, liquid L3 may be the same as the sealing liquid described above, or it may be different.
  • step (C3) Step of flowing emulsion into flow path and replacing lipophilic liquid with emulsion
  • emulsion E is injected from the injection port 221, and the emulsion E prepared in step (C1) is flowed into the flow path FC of the detection device 400, replacing the liquid L3 in the flow path FC with the emulsion E.
  • the excess liquid L3 is discharged from the flow path FC while the liquid L3 sealing the well W remains, and the flow path FC is filled with emulsion E.
  • a restriction enzyme can be added to the emulsion to cut the nucleic acid at specific sequences, releasing the target molecule, the nucleic acid, into the emulsion droplets.
  • the cells can be cultured in the emulsion for a certain period of time, causing the secretion, which is the target molecule, to be released into the emulsion droplets.
  • the contents of the cell can be dispersed within the well W by adding a reagent that dissolves the cell membrane to the buffer B in the well W.
  • the reagent that dissolves the cell membrane can be the anionic surfactant mentioned above.
  • step (C6) Step of detecting target molecules
  • a reaction between the target molecules diffused in the wells W and the detection reagent is caused to occur, and the target molecules are detected.
  • the detection reagent may be placed in the wells W in advance.
  • a detection kit having a detection device 400 configured as described above can detect target molecules released from a target substance in a solution with high sensitivity.
  • the above-described method for detecting target molecules also makes it possible to detect target molecules with high accuracy.
  • the method for detecting a target molecule of this embodiment will be described with reference to Figures 4 to 8 and 10.
  • the method for detecting a target molecule includes the steps of performing the following operations.
  • the above-mentioned detection device 100 is used to detect a target molecule contained in a sample in the second well W2 of the detection device 100.
  • (AA1) A step of adjusting the concentration of a liquid sample containing a target substance to obtain a sample solution;
  • (AA2) A step of sealing the sample solution in a first well independently using a sealing liquid;
  • (AA3) A step of obtaining an evaluation substance in the first well;
  • (AA4) A step of distributing one evaluation substance per second well;
  • (AA5) A step of detecting a target molecule.
  • step (AA1) A step of adjusting the concentration of a liquid sample containing a target substance to obtain a sample solution.
  • this step may be abbreviated as "step (AA1)".
  • each step may be abbreviated in the same manner.
  • Step (AA1) differs from step (A1) in the following respects, but is otherwise the same, so explanation will be omitted.
  • the sample may contain a surfactant, and examples of the surfactant include those described in the first embodiment.
  • the concentration of the surfactant is preferably 0.0011 g/L to 55.5 g/L (0.0001 to 5% by volume [v/v]) relative to the total volume of the sample, more preferably 0.0111 g/L to 22.2 g/L (0.001 to 2% by volume [v/v]), and even more preferably 0.111 g/L to 11.1 g/L (0.01 to 1% by volume [v/v]).
  • the surfactant concentration is 55.5 g/L or less relative to the total volume of the sample, it will have less adverse effects on the reaction in the subsequent detection of target molecules.
  • the concentration of the sample is adjusted based on the volume of the fluidic device used so that one target substance M1 is placed in each first well.
  • the concentration of the sample should be determined by conducting a preliminary experiment using the fluidic device.
  • a process for visualizing the target substance may be performed. By performing such a process, it is possible to extract only the first well, which contains only one target substance, in a later process and use it as the measurement subject. Extraction can be performed, for example, by taking a magnified image using a microscope and analyzing the obtained image using known detection software.
  • the target substance is a cell, it may be detected directly using a bright field image of a microscope, or the cells may be stained with a trypan blue solution or the like. If the target substance is a nucleic acid, it may be made to emit light using a known fluorescent staining method or a method such as an ICA reaction, and a well containing one molecule of nucleic acid may be identified based on the fluorescence intensity. If the target substance is a nucleic acid, for example, the nucleic acid may be cut with a restriction enzyme to obtain a nucleic acid fragment as the target molecule.
  • step (AA2) Step of sealing the sample solution in the first wells by sealing liquid
  • the sample solution L1 is filled into each of the first wells W1 independently.
  • this step may be abbreviated as “step (AA2)”.
  • the detection device 100 is divided into a first member 110 and a second member 120, and the first member 110 is used in this step.
  • the process of injecting sample solution L1 containing a detection reagent into internal space S1 from injection port 211 is the same as process (A2), so a description thereof will be omitted.
  • sealing liquid L2 is injected into the internal space S1 from the injection port 211.
  • sealing liquid L2 may be dripped onto the first wells W1 of the first plate 11 to seal each of the first wells W1, or the solution on the first wells W1 may be scraped off with a spatula or the like, and then sealing liquid L2 may be dripped onto the first wells W1 to separate each of the first wells W1.
  • the sealing liquid L2 may be the sealing liquid L2 described above, may be a lipophilic liquid, and may not contain a surfactant.
  • the sealing liquid L2 individually seals each of the multiple first wells W1, and the first wells W1 containing the sample solution L1 become independent reaction spaces.
  • the sample solution L1 replaced with the sealing liquid L2 and the excess sealing liquid L2 are discharged from the discharge port 212.
  • step (AA3) Step of Obtaining Evaluation Objects in First Wells
  • particulate evaluation objects containing target molecules are obtained from the target substance M1 distributed to each first well W1.
  • this step may be abbreviated as “step (AA3)”.
  • the method for obtaining an evaluation item from a target substance is selected depending on the type of target substance in question.
  • the target molecules to be evaluated may be released from the target substance, and the resulting target molecules may be captured by capture particles, which will be described later, to obtain an evaluation item.
  • the target substance is DNA or RNA
  • multiple fragments i.e., the target molecule
  • the target substance is DNA or RNA
  • multiple fragments may be obtained from a single fragment by ultrasonic disruption or enzymatic degradation.
  • the detection device may be heated to separate the individual nucleic acids through thermal denaturation.
  • the target substance is a protein aggregate
  • the aggregates may be loosened by ultrasonic irradiation, an enzyme that breaks down proteins may be used, or a reagent that breaks down protein aggregates may be added.
  • the cell may be disrupted by ultrasound or the cell membrane may be disrupted by applying electrical stimulation.
  • cells which are the target substance, may be cultured in the first well for a certain period of time, and secretions such as cytokines secreted by the cells may be detected as target molecules.
  • the capture particle has a particle and a site to which the target molecule specifically binds.
  • the particles are not particularly limited, and examples include polymer particles, magnetic particles (particles containing a magnetic material), glass particles, etc.
  • the particles are preferably surface-treated to avoid non-specific adsorption.
  • particles having functional groups such as carboxyl groups on the surface are preferred to immobilize specific binding substances. More specifically, products such as "Magnosphere LC300" manufactured by JSR Corporation can be used.
  • the site to which the target molecule specifically binds can be selected from the same group as the specific binding substances described above, depending on the type of target molecule to be captured.
  • the radius of the captured particles is preferably 0.5 ⁇ m or more and 2.5 ⁇ m or less, and more preferably 1 ⁇ m or more and 2 ⁇ m or less.
  • the density of the captured particles may be greater or less than the density of water, where the density of water at 20° C. is 1 g/ml (g/cm 3 ).
  • the density of the captured particles may be, for example, 0.7 g/cm 3 or more and 1.3 g/cm 3 or less, or 0.8 g/cm 3 or more and 1.2 g/cm 3 or less.
  • step (AA4) Step of distributing one molecule of the evaluation substance to each second well
  • the evaluation substance in the sample solution L1 is distributed to each second well W2, one molecule at a time.
  • this step may be abbreviated as “step (AA4)”.
  • Process (AA4) differs from process (A4) in the following respects, but is otherwise the same, so a description of it will be omitted.
  • the first well W1 and the second well W2 are connected as shown in FIG. 10, and the evaluation object E in the first well W1 moves to the second well W2.
  • the evaluation object E contains the target molecule M2.
  • T d1 / v ... (1)
  • T (unit: sec) is the contact time.
  • d1 (unit: ⁇ m) is the diameter of the evaluation object.
  • v (unit: ⁇ m/sec) is the migration speed of the evaluation substance in the sample solution from the first well to the second well)
  • the contact time T is preferably longer than the time represented by the following formula (1)-1.
  • T d2 / v ... (1) - 1 (d2 (unit: ⁇ m) is the distance from the bottom surface of the first well to the surface where the second well is formed when the surface where the first well is formed and the surface where the second well is formed are brought into contact with each other.)
  • the detection device 100 Before the formation surface of the first well is brought into contact with the formation surface of the second well, the detection device 100 may be left stationary and the evaluation object may be moved within the first well toward the formation surface of the first well.
  • the time for leaving the device stationary may also be set according to the depth of the first well W1 using the above-mentioned movement speed v. For example, if the depth of the first well W1 is H, the time for the evaluation object to move to the middle position in the depth direction of the first well W1 can be calculated as H/(2 ⁇ v).
  • the moving speed v in the above formula (1) is a speed represented by the following formula (2).
  • v [2 g r 2
  • g the gravitational acceleration (unit: cm/s 2 )
  • r the radius of the evaluation object (unit: cm)
  • ⁇ the density of the evaluation object (unit: g/cm 3 )
  • ⁇ s the density of the sample solution (unit: g/cm 3 )
  • ⁇ is the viscosity of the sample solution (unit: g/(cm ⁇ s)).
  • the density ⁇ s of the sample solution is the density at 20° C.
  • the density ⁇ s is 0.998 g/cm 3 , and 1 g/cm 3 is used as the approximate value.
  • the physical property values of the sample solution are those of the component with the largest volume ratio among the components that make up the sample solution.
  • an aqueous solution such as a buffer is used as the sample solution.
  • the physical property values of water are used as the physical property values of the sample solution.
  • r can be the radius of the capture particle, and ⁇ can be the density of the capture particle.
  • ⁇ is approximated to 1.05.
  • the short diameter of the cell is measured by taking a magnified image of the cell (magnification: 4-20 times) using a microscope and using image analysis software to measure the short diameter of the cell contained in the image obtained.
  • the "length of the short side" of the "smallest rectangle circumscribing the cell” is taken as the short diameter of the cell.
  • the short diameters of all cells contained in the captured image are measured, and the average value is used as the above r.
  • To calculate the average short diameter of cells measure the short diameters of 10 or more cells according to the method described above. If the number of cells in one enlarged image is less than 10, use multiple enlarged images, measure the short diameters of all cells in each enlarged image until the total number is 10 or more, and calculate the average short diameter.
  • the moving speed v in formula (1) is calculated by substituting the acceleration generated in the captured particle when a magnetic force is applied to the captured particle at the midpoint between the bottom of the first well W1 and the bottom of the second well W2, in place of g in formula (2) above.
  • the acceleration can be calculated by a known method from the magnetic force acting on the captured particle and the physical properties of the captured particle.
  • the evaluation object may be moved by applying a centrifugal force from the first well W1 side to the second well W2 side to the detection device.
  • the moving speed v in formula (1) is calculated by calculating the centrifugal acceleration from the operating conditions (rotation speed, rotation radius) of the centrifuge used, and using the calculated centrifugal acceleration instead of g in formula (2) above.
  • step (AA5) Step of detecting target molecules
  • the target molecules are reacted with the detection reagent to detect the target molecules contained in the evaluation object.
  • this step may be abbreviated as “step (AA5)”.
  • the detection reagent may be placed in the second well W2 in advance, or may be added later.
  • the subject to be evaluated is a cell, it is advisable to culture the cells in the second well W2 for a certain period of time to allow the cells to release the target molecule.
  • the detection method can be the method described in step (A5).
  • the above-described method for detecting target molecules makes it possible to detect target molecules with high accuracy.
  • step (AA1) is performed, but step (AA1) may be omitted. If step (AA1) is not performed, after filling the first wells with the sample solution, a first well W1 in which one target substance M1 is placed may be extracted from among the multiple first wells in step (AA3), and the subsequent steps (A4A) and (AA5) may be performed.
  • the detection device 100 shown in this embodiment is just an example, and detection devices with other configurations can also be used.
  • the detection method for detecting target molecules of this embodiment can be implemented using the detection device 200 described in the second embodiment.
  • [Method for detecting target molecules] 12 to 14 are explanatory diagrams of a method for detecting a target molecule using the detection device 200.
  • the detection device 200 first, the step (AA1) is carried out in the same manner as described above.
  • a sample solution L1 containing a detection reagent is injected into the internal space S (flow path FC) from the injection port 151, and the first well W1 that opens into the internal space S is filled with the sample solution L1.
  • the second well W2 is also filled with the sample solution L1.
  • a sealing liquid L2 containing a surfactant is injected into the internal space S (flow path FC) from the injection port 151.
  • the sealing liquid L2 flows in the internal space S in the planar direction of the well formation surface 11a, and washes away the sample solution L1 that is not contained in the first well W1 and the second well W2 in the internal space S, replacing the internal space S.
  • the sealing liquid L2 individually seals each of the multiple first wells W1 and second wells W2 (step (AA2)).
  • the sample solution L1 replaced with the sealing liquid L2 and the excess sealing liquid L2 are discharged from the discharge port 152.
  • steps (AA3) to (AA5) can be carried out according to the method described above to detect the target molecule.
  • the detection device 200 has an inlet 151 and an outlet 152 in the first plate 15, this is not limited to this.
  • the detection device 200 may have a first plate 11 and a second well plate 16 having an inlet 161 and an outlet 162.
  • the target molecule can be detected with high accuracy.
  • the detection device 100 according to the first embodiment described above was used as the detection device.
  • the first plate 11 and the second plate 12 were produced by injection molding.
  • the first lid member 21 and the second lid member 22 were produced by injection molding. Cycloolefin polymer was used as the forming material.
  • the opening diameter (longer diameter) of the first well was 50 ⁇ m, and the depth of the first well W1 was 50 ⁇ m.
  • the volume of each first well W1 was 98 pL.
  • the opening diameter (longer diameter) of the second well W2 was 10 ⁇ m, and the depth of the second well was 15 ⁇ m.
  • the volume of each second well W2 was 1100 fL.
  • the first plate 11 and the first lid member 21 were attached using double-sided tape (manufactured by Nitto Denko Corporation) to form the first member 110.
  • the second plate 12 and the second lid member 22 were attached using double-sided tape (model number: No. 5610BN, manufactured by Nitto Denko Corporation) to form the second member 120.
  • a cytokine capture antibody (Anti-Mouse IL-2 MAb (Clone JES6-1A12), model number: MAB702-100, R&D Systems, Inc.) was bound to magnetic beads (MS300/Carboxyl, manufactured by JSR Corporation).
  • Magnetic beads bound to cytokine capture antibodies were added to pure water to prepare a magnetic bead solution.
  • the magnetic beads bound to cytokine capture antibodies are the "capture particles.”
  • Example 1A Comparative Example 1A
  • the first member 110 was filled with pure water, and the second member 120 was filled with PBS-T (0.05% by volume of Tween 20).
  • the above-mentioned bead solution was added to the first member 110 so that the amount of capture particles introduced into the first member 110 became 4 ⁇ 10 6 particles.
  • FC-40 containing Tween 20 at a saturated concentration was used as the sealing liquid (sealing liquid 1).
  • FC-40 containing no Tween 20 was used as the sealing liquid (sealing liquid 2).
  • the first plate 11 was removed from the first member 110, and the second plate 12 was removed from the second member 120. 100 ⁇ L of sealing liquid (sealing liquid 1 in Example 1A, sealing liquid 2 in Comparative Example 1A) was added to each well formation surface (State A).
  • the detection device was divided into a first plate 11 and a second plate 12, and 100 ⁇ L of sealing liquid (sealing liquid 1 in Example 1A, sealing liquid 2 in Comparative Example 1A) was added to each well-forming surface.
  • the second lid member 22 was reattached to the second plate 12 to form a second member 120.
  • the first plate 11 and the second plate 12 in state A and the second plate 12 in state B were observed under a microscope.
  • the following equipment was used.
  • Figures 22 to 24 are enlarged photographs showing the results of the working example.
  • Figures 25 to 27 are enlarged photographs showing the results of the comparative example.
  • Figures 22 and 25 are enlarged photographs of the first plate 11 in state A
  • Figures 23 and 26 are enlarged photographs of the second plate 12 in state A
  • Figures 24 and 27 are enlarged photographs of the second plate 12 in state B.
  • Example 1A it was confirmed that beads were present in spots on the second plate 12 in state B, as shown in Figure 24. That is, in the example in which sealing liquid 1 containing a surfactant was used, it is believed that the beads sealed in the first well of the first plate 11 moved to the second well of the second plate 12, resulting in the beads moving in spots to the second well where the first well overlapped.
  • Example 1B Pure water was filled into the first member 110. Next, the above-mentioned magnetic bead solution was added so that the amount of captured particles in the first member 110 became 1 ⁇ 10 6 particles. Furthermore, 500 ⁇ L of FC-40 was filled as a sealing liquid.
  • the second member 120 was filled with 100 ⁇ L of pure water. Then, 200 ⁇ L of FC-40 was filled as a sealing liquid.
  • the first plate 11 was removed from the first member 110, and the second plate 12 was removed from the second member 120. 100 ⁇ L of sealing liquid was added to each well formation surface (state A).
  • the detection device was divided into a first plate 11 and a second plate 12, and 100 ⁇ L of sealing liquid was added to each well-forming surface.
  • the second lid material 22 was reattached to the second plate 12 to form a second member 120.
  • Experimental Example 2B Experimental Example 2 was carried out in the same manner as Experimental Example 1, except that the well-formed surface 11a of the first plate 11 and the well-formed surface 12a of the second plate 12 were bonded together by pressing them against each other for 10 seconds.
  • Experimental Example 3 was carried out in the same manner as Experimental Example 1, except that the well-formed surface 11a of the first plate 11 and the well-formed surface 12a of the second plate 12 were bonded together by pressing them against each other for three minutes.
  • Figures 28 to 32 are enlarged photographs showing the results of the examples.
  • Figure 28 is an enlarged photograph of the first plate 11 in state A
  • Figure 29 is an enlarged photograph of the second plate 12 in state A
  • Figures 30 to 32 are enlarged photographs of the second plate 12 in state B (Figure 30: Experimental Example 1B
  • Figure 31 Experimental Example 2B
  • Figure 32 Experimental Example 3B).
  • the density ⁇ of the trapped particles used was 1.1 g/cm 3 , and the particle radius r of the trapped particles was 1.5 ⁇ m (1.5 ⁇ 10 ⁇ 4 cm).
  • the density ⁇ s of pure water was 1 g/cm 3
  • the viscosity ⁇ was 1 mPa ⁇ s (0.01 g/(cm ⁇ s))
  • the gravitational acceleration was 9.8 ⁇ 10 2 cm/s 2
  • the settling velocity of the trapped particles was calculated to be 0.49 ⁇ m/s from the following formula (2).
  • v [2 g r 2
  • the present invention provides a detection kit that enables highly sensitive detection of target molecules released from a target substance in a solution. It also provides a method for detecting target molecules using such a detection kit.

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Abstract

Ce kit de détection comporte un dispositif de détection de molécule cible et un liquide d'étanchéité qui est utilisé dans le dispositif de détection, le liquide d'étanchéité consistant en un mélange d'un liquide lipophile et d'un tensioactif, la concentration en tensioactif dans le liquide d'étanchéité étant située dans la plage allant de 1 à 100 % en volume de la concentration de saturation du tensioactif dans le liquide d'étanchéité, le dispositif de détection comprenant une première plaque de puits comportant une pluralité de premiers puits dans une surface de celle-ci, et une seconde plaque de puits comportant une pluralité de seconds puits dans une surface de celle-ci, la première plaque de puits et la seconde plaque de puits étant agencées de telle sorte que les premiers puits et les seconds puits se fassent face, un canal d'écoulement à travers lequel un fluide s'écoule étant formé entre la première plaque de puits et la seconde plaque de puits, et les premiers puits étant superposés sur les seconds puits dans une vue en plan.
PCT/JP2023/037861 2022-10-20 2023-10-19 Kit de détection et procédé de détection de molécule cible WO2024085226A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011506998A (ja) * 2007-12-17 2011-03-03 ハイ−チング ゴング マイクロ流体デバイス
WO2015115635A1 (fr) * 2014-01-31 2015-08-06 凸版印刷株式会社 Kit d'analyse de biomolécule et procédé d'analyse de biomolécule
WO2017043530A1 (fr) * 2015-09-08 2017-03-16 凸版印刷株式会社 Procédé de détection d'une substance biologique
JP2018031631A (ja) * 2016-08-23 2018-03-01 パナソニックＩｐマネジメント株式会社 マイクロ流体チップ、微生物検出装置及び微生物検出方法
WO2019098301A1 (fr) * 2017-11-17 2019-05-23 凸版印刷株式会社 Procédé de détection de molécules cibles
JP2022159046A (ja) * 2021-03-31 2022-10-17 東レ株式会社 分析用チップ、分析方法及び分析用チップの製造方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011506998A (ja) * 2007-12-17 2011-03-03 ハイ−チング ゴング マイクロ流体デバイス
WO2015115635A1 (fr) * 2014-01-31 2015-08-06 凸版印刷株式会社 Kit d'analyse de biomolécule et procédé d'analyse de biomolécule
WO2017043530A1 (fr) * 2015-09-08 2017-03-16 凸版印刷株式会社 Procédé de détection d'une substance biologique
JP2018031631A (ja) * 2016-08-23 2018-03-01 パナソニックＩｐマネジメント株式会社 マイクロ流体チップ、微生物検出装置及び微生物検出方法
WO2019098301A1 (fr) * 2017-11-17 2019-05-23 凸版印刷株式会社 Procédé de détection de molécules cibles
JP2022159046A (ja) * 2021-03-31 2022-10-17 東レ株式会社 分析用チップ、分析方法及び分析用チップの製造方法

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